The identification of genetic risk variants that may predispose certain patients to disease requires analysis of germline DNA from affected cases. In this study, we demonstrate that the Internet can provide a secure, confidential, and convenient way to bolster accrual of target patients from across the country.
Our protocol had a similar design to the Harvard Myeloproliferative Disorders Study. That protocol used the Internet to collect blood from 345 participants over a 1-year period (i.e. 0.41%/month of the 7,000 newly diagnosed cases of myeloproliferative disorders in the U.S.) [22
]. We had hoped to accrue patients at a similar rate. However, our recruitment was significantly lower, as we only recruited 45 patients over an 8-month period (i.e. 0.11%/month of the 14,000 estimated newly diagnosed cases over the 8 month-period). Limiting factors for enrollment may include an older age at onset of lung cancer and differences in socio-economic characteristics compared to those who develop myeloproliferative diseases [23
]; these factors ultimately may reduce these patients’ familiarity/comfort/access to the Internet. Socioeconomic factors have previously been reported to significantly influence the completion of an Internet-based behavioral study in smokers [24
] and may also exist in never smokers [25
]. Second, disease-free survival in NSCLC is far shorter than in myeloproliferative disorders, limiting the extra time and effort patients are willing to allot to research. This reason was given by several patients who received screening documents but did not return them. Interestingly, the majority of patients who signed the consent form ultimately completed the questionnaire, had their blood drawn, and sent the blood specimens. This success rate suggests that the study participation was not overly complicated and that a more significant limitation of the study was awareness. Thus, in the future, we will expand efforts to connect with individuals appropriate to this study, through patients’ groups, development of collaboration with other institutions, and presentation of the study at oncology meetings.
While somatic EGFR
T790M mutations are common in patients with acquired resistance to EGFR inhibitors [16
], germline EGFR
T790M mutations are rare. This variant was initially reported in 2005 in a family of European descent, in which 6 family members in 3 generations had lung cancer [26
]. We found the germline EGFR
T790M variant in 2 of 369 cases (0.54%) of never smokers with NSCLC; both patients had family histories significant for lung cancer. In a separate study, no germline mutation was identified in a series of 237 individuals with 3 or more first-degree relatives with lung cancer and 32 patients with bronchioloalveolar carcinoma [27
]. One germline EGFR
T790M mutation was identified in a cohort of 240 patients with previously untreated lung adenocarcinoma [28
]. Including our 2 patients, a total of 5 patients have now been reported to have the germline EGFR
T790M variant (). In these patients, lung cancer was diagnosed from age 50 to 72 years and was metastatic to the lung or other locations at the time of diagnosis. At least three patients were never smokers. Histology was adenocarcinoma and/or bronchioloalveolar carcinoma in all cases. EGFR
was genotyped in all the 5 germline EGFR
T790M cases. Somatic EGFR
activating mutations, which are known to be more frequent in never smokers with lung adenocarcinoma [6
], were identified in four of the five patients. Collectively, these data indicate that a germline T790M alteration is a genetic risk variant for lung cancer in never smokers. However, tumors develop only after a relatively long latency, suggesting other genetic alterations, such as additional EGFR
mutations, may collaborate with T790M [29
Lung cancer patients with germline T790M mutations
We were unable to confirm a reported association between rs763317 in the EGFR
gene and lung cancer [17
]. In the previous report, female never smokers with lung adenocarcinoma heterozygous at the locus had a 1.2-fold increased risk of lung cancer relative to individuals homozygous for the G allele. Individuals homozygous for the A allele were 3.6 times more likely to develop lung adenocarcinoma. Based on this effect size and the frequency of this SNP in our control population, we estimated 82% power to detect this association in our Asian cohort, and 96% power to detect the association in our white cohort. Therefore, our failure to replicate this association is not due to low power. One possible explanation is that this association was a false positive. Alternatively, the initial report could have over-estimated the effect size at this locus, due to the "winner's curse," and therefore our power to replicate the association is lower than we calculated.
We were also unable to replicate the previous reported association between SNPs rs8034191 and rs1051730 in CHRNA3
and lung cancer risk [13
]. Unlike EGFR
rs763317, these associations are much more modest. Moreover, the minor allele frequency in Asian populations at these SNPs is much lower than in the European populations in which the association was first discovered. Our power to detect an association in our cohort, therefore, was limited. However, as it has been suggested that these SNPs affect smoking behavior as well as lung cancer risk, and as we focused on never smokers with lung cancers, we cannot discount the possibility that the SNP only has an effect in smokers (or influences smoking behavior) and is truly not associated with lung cancer in never smokers.
To conclude, this study shows the feasibility of bolstering accrual for germline predisposition studies in never smokers with lung cancer by collecting clinical information and blood sample specimens from appropriate patients throughout the nation using an Internet-based protocol. We plan in future studies to conduct genome-wide association studies with more cases and controls to identify genetic risk variants in this disease population.
STATEMENT OF TRANSLATIONAL RELEVANCE
This study examines in US and Japanese never smokers with lung cancer 3 genetic variants reported to be associated with genetic susceptibility to the disease. We collected germline DNA from a large cohort of American and Japanese never smokers with or without lung cancer. We used a dedicated internet-based protocol to bolster accrual from patients throughout the U.S. Contrary to previous studies including smokers, no differences were observed in EGFR rs763317 or CHRNA3 (rs8034191/rs1051730) frequencies between cases and controls. The CHRNA3 loci are likely to be associated with smoking addiction rather than the development of lung cancer. We did identify a germline EGFR T790M variant in 2 of 369 (0.54% of cases). This study validates the EGFR T790M as a rare lung cancer risk variant in never smokers and illustrates the usefulness of the Internet in recruiting patients throughout the country for minimal risk studies.